The invention relates to a device and method for screening molecularly diverse chemical mixtures such as combinatorial chemical libraries and natural products for substances which bind with affinity and specificity for a macromolecule (a receptor) or complex thereof. The device employs ultrafiltration of compounds in solution to identify, concentrate and isolate candidate compounds that bind to the receptor.
The selection and identification of pharmacologically active molecules directly from compound mixtures called combinatorial libraries is an approach to drug discovery and lead optimization that is cost effective compared to testing individual molecules singly.
Small organic molecules that possess unique spatial and thermodynamic properties which allow them to bind specifically to biologically related macromolecules and their complexes are candidates for therapeutic drugs. Examples of the macromolecules include proteins, DNA, RNA, viral particles and whole cells. The binding of these compounds to such macromolecules can transiently or permanently eliminate or activate the function of those macromolecules. Thus, a compound which binds to and eliminates the function of a coat protein of a virus could serve as an effective pharmacologic agent against that virus. Similarly, a compound which binds to and activates a receptor for estrogen could serve as a useful active agent for estrogen replacement therapy.
In the classical approach to drug development, single compounds are individually synthesized and tested for a given biological activity. Consequently, a one-to-one correlation between the single chemical structure and biological activity is maintained. However, because both the synthesis and bioassays are very labor intensive, this approach is inefficient and expensive. The pharmaceutical industry has recognized the significant enhancement in efficiency that may be gained by simultaneously producing many compounds, called chemical libraries, and testing them as a group. Numerous methods have been developed for the preparation of complex chemical libraries containing considerable molecular diversity (Moos, 1993). A difficult challenge has been the screening of these libraries to detect molecules with a desired biological activity or binding profile that is specific for a given target receptor. The difficulty comes from the fact that although complex mixtures can be assayed and a given biological activity or binding can be determined to be present in the mixture, the identity of the molecule(s) responsible for the given activity or binding is not known.
An essential aspect of screening combinatorial libraries is the ability to identify the active components in these large mixtures, usually based on the strength of binding to a selected macromolecule. Common approaches to this problem utilize a) immobilization of either the target or library molecules on a solid phase support to facilitate identification of the active compounds in the library, or b) iterative resynthesis of components of the library until a single active compound is identified. However, immobilization alters the affinity characteristics of the bound species, and the resynthesis approach is labor intensive and time consuming.
One strategy to identifying drug candidates has been to prepare the chemical compounds in a library covalently linked to specific areas on a surface. In this technique, the use of photochemically labile protecting groups allows the light-directed, spatially addressable, parallel, chemical synthesis of thousands of compounds at defined sites on a microchip. (McGall, 1995; Stryer, 1991) Compounds which bind a specific receptor are then identified by bathing the chip with a solution of the receptor and, through various means, identifying the location of the bound macromolecule on the chip. The structure of the compound which binds the given macromolecule is identified by location on the chip. Similar approaches synthesize compounds bound to individual pins or beads, which are then assayed by evaluating which bead or pin binds the macromolecule. (Lam et al., 1991; Geysen, H. M. et al., 1984) Molecular biological approaches include having a compound, typically a peptide, tethered to a surface, i.e. a phage particle. (Smith, Science, 1985)
The majority of the available methods to screen drug candidates require the compounds under investigation to be covalently linked to the given surface in order to define the relationship between structure and activity or affinity for the macromolecule. It is well understood in the industry that such attachments chemically alter the compounds and can result in both false positive as well as false negative results. Consequently, these approaches are of limited use.
Alternatively, for assay purposes, individual compounds can be released into solution from the macromolecular surfaces on which they are bound. Several coding methods have been developed to relate chemical structure to a particular surface from which a compound has been released. However, all of these strategies remain labor intensive.
A general approach to reducing the number of analyses which must be preformed to relate a particular physical property, e.g. spectral property, biological activity, to a given chemical structure uses indexed libraries (referred to in the more general sense as sample multiplexing). This technique can be used to help reduce the number of assays which must be performed in screening libraries by any of the present methods. This approach involves preparing two series of sublibraries. (Smith et al., 1994; Pirrung, 1995) The sublibraries are tested as a matrix whose column and row intersections serve as indices for a unique structure in the sublibraries. (Woodbury et al., 1995) Although this approach reduces the labor in bioassaying the compounds, the number of assays (equal to 2.sqroot.n where n is equal to the number of compounds in the libraries) remains large for large libraries. Further, when more than one compound in the library is active, such indexing may not give a unique solution to the relationship of structure and activity.
A major obstacle toward identifying new candidates for drug development is that, although many methods have been developed to prepare mixtures of chemically diverse compounds, the screening of these mixtures is inefficient and labor intensive. There is, therefore, a need for methods (assays) which allow for rapid and efficient screening of large numbers of compounds for those having specific binding characteristics. A useful system would provide for the screening of compounds for binding to macromolecules and their complexes in which there is no need to bind to a surface. Another goal is to preserve quantities of difficult to obtain macromolecules such as recombinant proteins. The system should include conditions where structural information on the bound specific compound is obtainable through operation of the assay.